Human mesenchymal stem cells (MSCs) are multipotent stem cells that differentiate into many of the cells resident in musculoskeletal and stromal tissues of the human body, including fibroblasts, chondrocytes, osteoblasts, myocytes, and adipocytes. While differentiation of the MSCs into appropriate lineages may enhance healing of injured tissues, inappropriate lineage specification may be responsible for numerous pathophysiologic processes, including the decreased bone and increased fat in osteoporotic bones, and the calcification of atherosclerotic vessel walls. Regulation of the lineage commitment of MSCs by local microenvironmental cues therefore may be critical to our fundamental understanding of numerous degenerative as well as healing processes. The long term objective of this research is to characterize the cues within the local surrounding microenvironment that drive the lineage specification and differentiation of human mesenchymal stem cells (MSCs), and the molecular pathways involved. The investigator has recently discovered that adhesion of MSCs to fibronectin regulates a commitment switch in the MSCs between adipogenic and osteogenic lineage specification, through a mechanism involving RhoA signaling and cytoskeletal tension. In this proposal, the investigator proposes to further characterize how adhesive and mechanical cues regulate the commitment of MSCs to osteogenic and adipogenic lineages. Specific Aim 1 will be to investigate the role of integrin-mediated cell adhesion in modulating mesenchymal stem cell commitment by characterizing the binding interactions that drive the MSC lineage commitment switch between osteoblasts and adipocytes. Specific Aim 2 will be to investigate the coordination of RhoA and cytoskeletal tension in the regulation of stem cell commitment. Specific Aim 3 will be to investigate the ability of RhoA to regulate stem cell commitment and differentiation in an animal model. Together, these studies will define roles of cell adhesion, RhoA, and cytoskeletal tension in MSC lineage commitment, and establish a molecular basis for the regulation of MSC differentiation by microenvironmental cues.